Hot start reverse transcription by primer design
View Patent ↗The present teachings provide methods, compositions, and kits for performing primer extension reactions. In some embodiments, a reverse transcription reaction is performed on a target polynucleotide with a hot start primer comprising a blunt-ended self-complementary stem, and a loop, and extension products form at high temperatures but reduce extension product formation at low temperatures.
1. A composition comprising an RNA target, a reverse transcriptase, and a hot start reverse transcriptase (RT) primer, wherein the hot start RT primer comprises a sequence that forms a loop and a self-complementary stem at a temperature of 27° C. or lower, wherein the self-complementary stem, when formed, comprises a target-specific region hybridized to a quencher region, wherein the target-specific region is substantially unhybridized with the quencher region in the self-complementary stem at a temperature between 35° C. to 60° C., and wherein the target-specific region is hybridized with the RNA target.
2. The composition according to claim 1 wherein the RNA target is a messenger RNA.
3. The composition according to claim 1 wherein the RNA target is a small non-coding RNA.
4. The composition according to claim 3 wherein the small non-coding RNA is a micro RNA.
5. The composition according to claim 1 wherein the loop comprises 8-24 nucleotides.
6. The composition according to claim 1 wherein the self-complementary stem comprises 6-12 nucleotide base-pairs.
7. The composition according to claim 1 wherein the target-specific region further comprises at least one nucleotide present in the loop.
8. The composition according to claim 1 wherein the target-specific region, when hybridized to the quencher region, forms a blunt-ended structure.
9. A kit for reducing primer extension products at a low temperature and allowing primer extension at a high temperature comprising;
a hot start reverse transcriptase (RT) primer, a reverse transcriptase, a buffer, and dNTPs, wherein the hot start RT primer comprises a sequence that forms a loop and a self-complementary stem at a temperature of 27° C. or lower, wherein the self-complementary stem, when formed, comprises a target-specific region hybridized to a quencher region, wherein the target-specific region is complementary to an RNA target, and wherein the target-specific region when hybridized to the quencher region forms a blunt-ended structure, a structure with a one-nucleotide overhang, or a structure with a two-nucleotide overhang.
10. The kit according to claim 9 wherein the loop comprises 8-24 nucleotides.
11. The kit according to claim 9 wherein the self-complementary stem comprises 6-12 nucleotide base-pairs.
12. The kit according to claim 9 further comprising a second primer that selectively hybridizes to a DNA target and reagents for performing a polymerase chain reaction (PCR).
13. The kit according to claim 12 wherein the reagents for performing the PCR are included in a vessel that is the same vessel that contains at least one of the hot start RT primer, the reverse transcriptase, the buffer, and the dNTPs.
14. The kit according to claim 9 wherein the RNA target is selected from the group comprising a messenger RNA, a small non-coding RNA, and a micro RNA.
15. The composition according to claim 1 wherein the target-specific region comprises at least six nucleotides.
16. The kit according to claim 9 wherein the target-specific region comprises at least six nucleotides.
17. The kit according to claim 12 wherein the primer that selectively hybridizes to a DNA target is a hot start primer.
18. The kit of claim 9 , wherein the one-nucleotide overhang or the two-nucleotide overhang is at the 5′ end of the hot start RT primer.
19. The kit of claim 9 , wherein the one-nucleotide overhang or the two-nucleotide overhang is at the 3′ end of the hot start RT primer.